Identification and Characterization of CYP6 Family Genes from the Oriental Fruit Moth (Grapholita molesta) and Their Responses to Insecticides

Abstract

Cytochrome P450 (CYP) monooxygenases comprise a superfamily of proteins that detoxify xenobiotics and plant secondary metabolites in insects. The CYP6 family is unique to the class Insecta, and its members participate in the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of eight CYP6 family genes from Grapholita molesta (Busck), a global pest of pome fruits. P450 genes with the exception of CYP6AN35, which was most highly expressed in adults, consistently showed high expression in third- or fourth-instar larvae. The analysis of different tissues of adults showed that most of these genes were predominantly expressed in the midgut, Malpighian tubules, and/or fat body. The expression of these eight CYP6 genes was differentially affected by three representative insecticides: malathion (organophosphate), deltamethrin (pyrethroid), and chlorantraniliprole (carbamate). All eight CYP6 genes responded to malathion treatment. Only three CYP6 genes were highly expressed in deltamethrin-treated individuals. Chlorantraniliprole treatment exerted weak effects on gene expression. Interestingly, CYP6AN35 was a highly expression level in the adult head and its expression was induced by all three insecticides. CYP6AN35 may be a key gene in the metabolism of insecticides. This study provides a fundamental understanding of the functions of the CYP6 gene family in insecticide metabolism in G. molesta.

Keywords: Grapholita molesta; chlorantraniliprole; cytochrome P450 monooxygenases; deltamethrin; expression; malathion.

Figure 1

Predicted tertiary structures of eight CYP6 genes from G. molesta. Image colored by rainbow N-C terminus. (Supplementary File S1).

Figure 2

Phylogenetic analysis of eight CYP6 genes in G. molesta and B. mori CYPs. Forty-three CYP sequences from B. mori were used to construct this phylogenetic tree. The eight G. molesta CYPs are indicated by “●”.

Figure 3

Expression of eight CYP6 genes at different developmental stages of G. molesta Note: egg (Egg), first-instar larvae (1st), second-instar larvae (2nd), third-instar larvae (3rd), fourth-instar larvae (4th), fifth-instar larvae (5th), pupae (Pu), and adults (Ad). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey’s HSD test.

Figure 4

Expression patterns of eight CYP6 genes in different tissues of G. molesta. Note: head (HE), foregut (FG), midgut (MG), hindgut (HG), Malpighian tubule (MT), fat body (FB), spermary (SP), and ovary (OV). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey’s HSD test.

Figure 5

Relative expression of CYP6 genes in G. molesta treated with different concentrations of malathion. Note: control (CK), lethal concentration 10% (LC₁₀), lethal concentration 30% (LC₃₀), and lethal concentration 50% (LC₅₀). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey’s HSD test.

Figure 6

Relative expression of CYP6 genes in G. molesta treated with different concentrations of deltamethrin. Note: control (CK), lethal concentration 10% (LC₁₀), lethal concentration 30% (LC₃₀), and lethal concentration 50% (LC₅₀). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey’s HSD test.

Figure 7

Relative expression of CYP6 genes in G. molesta treated with different concentrations of chlorantraniliprole. Note: control (CK), lethal concentration 10% (LC₁₀), lethal concentration 30% (LC₃₀), and lethal concentration 50% (LC₅₀). Each treatment included three biological replicates. The data in the bar charts are presented as the means ± SEs. Different letters indicate significant differences at the p < 0.05 level, as determined by ANOVA and Tukey’s HSD test.